NL9401140A - Production of oligosaccharides in transgenic plants - Google Patents

Abstract

The invention relates to a process for producing oligosaccharides, comprising the steps of: selecting a gene which codes for an enzyme which is able to convert sucrose into an oligosaccharide; linking the gene to suitable transcription-initiation and transcription- termination signals to provide an expression construct; transforming a suitable plant cell by means of the expression construct; regenerating a transgenic plant from the transformed plant cell; growing the transgenic plant under conditions which enable the expression and activity of the enzyme; and isolating the oligosaccharides from the transgenic plant. The invention further relates to the product obtained by means of the process and the use thereof, and to transgenic plants and parts thereof which are able to produce oligosaccharides.

Description

PRODUCTION OF OLIGOBACCHARIDES XM TRANSGENIC PLANTS

The present invention relates to a process for producing oligosaccharides, to the oligosaccharides so produced, to transgenic plants and plant cells capable of producing oligosaccharides and to the uses of the oligosaccharides thus obtained.

In the food industry, there is an increasing trend towards "light" and low calorie. The use of too much fat and / or sugar in products is thereby avoided. In order to be able to provide a sweet taste to foods, more and more sugar substitutes are coming onto the market. Aspartame is a well-known example of this. Aspartame, however, has poor organoleptic properties.

Another type of sugar substitute is oligosaccharides. Oligosaccharides are molecules consisting of two or more monosaccharides, such as fructose and / or glucose. The monosaccharides in said oligosaccharides are linked together either by 6- (2-1) or by 6- (2-6) bonds. The number of monosaccharides in an oligosaccharide is indicated by the DP (Degree of Polymerisation) value. A DP value of 3 means that the oligosaccharide is made up of three monosaccharides. Oligofructoses are oligosaccharides, which consist entirely of fructose units. When an oligosaccharide also comprises one or more glucose units, they will be linked to a fructose unit by means of an o (1-2) bond. The composition of oligosaccharides is also referred to by the formula GJFn, where G is glucose and F is fructose and m is equal to 0 or 1, and n is an integer greater than or equal to o. Particularly suitable oligosaccharides are those in which m equals 1 and n is 2 to 8, preferably 2 or 3.

Oligosaccharides cannot or hardly be hydrolysed in the human stomach and small intestine. Oligofructose is known to have no effect on human digestive enzymes on the 6 (2-1) and 6 (2-6) bond in the molecule. As a result, they pass through the stomach and small intestine without being broken down and absorbed into the body. However, the oligosaccharides do not leave the body but are metabolized by the microbial flora of the colon. In addition to gas, volatile fatty acids are also released, which in turn serve as an energy source for the intestinal flora. This phenomenon explains why oligosaccharides have a lower energy value to humans than free sugars, such as glucose, fructose and sucrose, which are absorbed into the body. However, oligosaccharides do have enough sweetening power to serve as a sugar substitute.

Furthermore, oligosaccharides, in particular oligofructose, are known to have a bifidogenic effect, ie they stimulate the growth of Bifidobacteria in the digestive system. Bifidobacteria protect against the development of pathogenic bacteria and therefore have a positive effect on health. In addition, oligosaccharides are dietary fiber.

There are currently several oligosaccharides on the market, which are prepared in various ways.

Oligosaccharides can be made by the partial enzymatic hydrolysis of longer vegetable inulin chains. A method for this is described, for example, in European patent application 440,074.

Oligosaccharides can also be synthesized enzymatically. This enzymatic production path uses enzymes, fructosyl transferases, which convert sucrose to a mixture of oligosaccharides, and which are isolated from various microorganisms (JP-80/40193).

However, the known production routes have a number of drawbacks. Firstly, the two known production methods are relatively expensive. Furthermore, in addition to the desired oligosaccharides with a chain length of 2 to about 7, in the produced mixture there are also relatively many free sugars and oligosaccharides with a higher chain length.

The disadvantage of many free sugars is that the energy value of the mixture increases as a result. For example, free sugars have an energy content of 17 kilo joules per gram, while pure GF2 and GF3 have an energy content of 4 to 6 kilojoules per gram. Free sugars also cause tooth decay (caries).

Oligosaccharides with too high a chain length, on the other hand, have too little sweetness, which decreases the average sweetness of the mixture.

Unlike some other sweeteners, such as Aspartame, oligosaccharides have good organoleptic properties.

The aim of the present invention is to provide an alternative production route for oligosaccharides, which avoids the above drawbacks.

To this end, the invention provides a method of producing oligosaccharides, comprising the steps of: a) selecting a gene encoding an enzyme capable of converting sucrose into an oligosaccharide; b) linking the gene to appropriate transcription initiation and transcription termination signals to provide an expression construct; c) transforming a suitable plant cell with the expression construct; d) regenerating a transgenic plant from the transformed plant cell; e) cultivating the transgenic plant under conditions that allow for the expression and activity of the enzyme; and f) isolating the oligosaccharides from the transgenic plant.

The invention therefore provides a method by which an oligosaccharide or a mixture of oligosaccharides can be produced by means of transgenic plants or plant cells, which have more desirable properties in comparison with the industrially prepared industrial means.

The advantage of the method according to the invention is that the chain length distribution is narrower, as a result of which little or no free sugars occur in the end product. The consequence of this is a lower cariogenicity and the desired lower energy value. There are also fewer oligosaccharides with a chain length of more than 5. The advantage of this is that the oligosaccharides produced according to the invention have a higher specific sweetening power. The sweetening power depends on the “average chain length”. The higher the average chain length of a mixture, the lower the sweetening power. The advantage of a high specific sweetening power is that hardly any additional sweeteners need to be added when processing the product.

Something similar applies to solubility. Here, too, the solubility decreases with an increase in the average chain length. The mixtures according to the invention therefore have a higher solubility than the mixtures obtained by enzymatic synthesis or enzymatic hydrolysis. In addition, production costs are significantly reduced.

There are indications that short chains can be better absorbed into the bacterial body of Bifidus than long ones. The oligosaccharide mixtures produced by the method of the invention will therefore have a higher bifidogenic effect.

For the selection of a gene encoding an enzyme capable of converting sucrose into an oligosac charide, a search can be made in any possible organism containing fructosyl transferase activity, for example microorganisms, such as bacteria, or plants. Many microorganisms are known to contain fructosyl transferases that have the ability to produce fructans from sucrose. These enzymes transfer fructose units from sucrose to a fructan acceptor molecule. Usually microbial fructosyl transferases produce high DP fructans. The use of a number of fructosyl transferases for the production of transgenic plants for the production of such polysaccharides has already been described in the literature. It is known, for example, that the fructan pattern of those plants can be modified by the incorporation of the SacBen of Bacillus subtilis in plants (WO 89/12386). However, this always concerns the production of high molecular polysaccharides.

Another gene known to encode a fructosyl transferase, which can convert sucrose into high molecular fructans is the ftf gene of Streptococcus mutans. However, according to the present invention, it has now surprisingly been found that this fructosyl transferase produces important amounts of oligosaccharides in the trisaccharide class (1-kestose) in addition to high molecular fructans. In addition, mutants have been found that only accumulate trisaccharides and no polysaccharides.

Furthermore, mutants of the SacBaen of Bacillus subtilis are known which also mainly produce trisaccharides.

A large number of other microorganisms are also capable of fructosyl transferase production. These include but are not limited to endospore-forming rod bacteria and cocci (eg Bacillus), gram positive cocci (eg Streptococcus), gram negative aerobic rod bacteria, and cocci (eg Pseudomonas. Xantho monas. Azotobacter) gram negative optional anaerobic rod bacteria (e.g. Erwinia. Zvmomonas), actinomycetes (e.g. Actinomvces. Rothia) and cyanobacteria (e.g. Tolvpothrix tenuis).

The genes encoding these fructosyltransfer varieties may optionally be modified by targeted or random mutagenesis techniques to provide enzymes which have the desired oligosaccharide synthesizing enzymatic properties.

Bacterial fructosyl transferases have a relatively low KM for sucrose, about 20 mM. Sucrose concentrations in most plants are considerably higher and therefore these enzymes will also be active in plants. An important property of bacterial fructosyl transferase is their activity at low temperatures down to 0 ° C. Plants often come into contact with these temperatures, but even under those conditions the bacterial enzymes will still be active.

Fructosyl transferases can also be of vegetable origin. In plants, the biosynthesis and degradation of fructan only occurs in a limited number of species. Examples are the Asteraceae, Liliaceae and Poaceae families. Starting from the known vegetable fructosyltransferases, the genes suitable for the present invention can be isolated or produced either by targeted or random mutagenesis or by selection of already naturally occurring mutants.

An example of a very suitable vegetable fructosyl transferase is the sucrose sucrose fructosyl transferase (SST) which occurs in various plants and in particular catalyses the synthesis of trisaccharides.

For expression in plants of the selected fructosyl transferase gene, transcription initiation signals, such as promoters, enhancers and the like, may be added to the gene to obtain the desired expression construct. Such expression promoters can be specific for a special cell type or can be active in a wide variety of cell types. In addition, the time and place of expression can be determined using, for example, development-specific promoters. A commonly used promoter for gene expression in plants is the 35S Cauliflower Mosaic Virus Promoter (CaMV promoter) which is active in many cell types in the plant regardless of the stage of development of the plant. When the fructosyl transferase gene is from a plant, it is also possible to use its own regulatory sequences.

The preferred promoter may be a tissue specific or constitutive promoter, strong or weak depending on target plant and target. Examples of suitable promoters are the "sink" -specific patatin promoter and the potato-bound starch synthase promoter of the potato, or the sporamine promoter of the sweet potato.

To further increase transcription levels, the promoter may be modified and contain an enhancer duplication.

The translation of the mRNAs can be improved by adding a translational enhancer, such as the Alfalfa Mosaic Virus RNA4 translation enhancer signal, which must be present in the transcribed 5 'untranslated region.

For proper termination of transcription, a terminator sequence may be added to the constructs. An example of such a sequence is the nopaline synthase gene termination sequence.

Of course, the choice of expression signals suitable for a specific situation is within the reach of the average skilled person, without any further inventive work having to be done for this.

Sucrose, the substrate for the fructosyltransfer varieties, is a carbohydrate present in many different locations. It is synthesized in the cytoplasm and in addition, significant amounts can be found in cytosol, vacuole and the extracellular space (the apo-plast) or possibly other sites.

Since biochemical processes in plant cells are also often limited to a single or a number of cellular compartments, it is desirable to cause the accumulation of the products of the newly introduced genes to take place in a specific compartment. To this end, targeting sequences specific for cellular compartments may be present in the expression construct near the coding portion of the fructosyl transferase genes expressed in the transgenic plants.

Specific amino acid regions for targeting to the different cellular locations have previously been identified and analyzed. These DNA sequences can be linked to the fructosyl transferase genes such that the enzymatic activity is directed to a desired compartment of the cell or plant.

Thus, in a preferred embodiment of the invention, the expression construct also includes a targe tting sequence for targeting the fructosyl transferase activity to one or more specific plant cell compartments. Examples of preferred targeting are the signal sequence and vacuolar targeting sequence of the carboxy peptidase Y fcovi gene, that of potato patatin or that of sweet potato sporamine, or the signal sequence and apoplastic targeting sequence of the pathogenesis-related protein S gene fpr-sl. These are examples, however, and those skilled in the art will be able to select other targeting sequences.

In principle, the expression construct can be modified such that targeting takes place to any cell compartment, such as the vacuole, plastids, cell wall, cytoplasm etc.

It is often beneficial for the plant to control not only the location but also the time of expression of the introduced genes. For example, it is usually desirable to limit the expression of the newly introduced enzymatic activities to specific parts of the plant, for example, harvestable organs such as tubers, fruits or seeds. In addition, it is often desirable to initiate expression in these organs at a particular stage of development. This is certainly the case when the expression of the introduced genes interferes with normal development of such organs.

The oligosaccharides of the invention can be used as a substitute for sugar, corn syrup and isoglucose in nlightn versions of various foods. Examples of foods are sweets, biscuits, pastries, dairy products, baby food, ice cream and other desserts, chocolate and the like. The stimulation of Bifidobacteria is also important for animal health. The oligosaccharides according to the invention can therefore also be used, for example, in animal feed.

The present invention will be further elucidated by the examples below, which are only given to illustrate the invention and do not intend to limit it in any way. In the examples, reference is made to the accompanying figures which show the following:

Figure 1 shows the oligosaccharide producing activity of wild type and modified forms of the Streptococcus mutans fructosyl transferase fftfl incubated with sucrose and analyzed on TLC as described by Cairns, A.J. and Pollock, C.J., New Phytol. 109, 399-405 (1988). Samples of cultures derived from colonies and purified proteins were incubated overnight with 200 mM sucrose in 50 mM sodium phosphate buffer with 1% Triton X-100 at 37 ° C. Lane 1 shows the reaction products of one. mutans culture: lane 2 shows the activity of the purified enzyme from £. mutans: lane 3 shows the activity of a £. coli support carrying the plasmid pTS102; lane 4 shows the activity of a £. coli strain harboring plasmid pTD2; lane 5 shows the activity of a £. coli cell transformed with the mature £. mutans fructo-syl transferase gene under the regulation of a £. coli promoter. The oligosaccharide standards used are in lane A an extract of an Allium cepa bulb, and in lane H an extract of a Helianthus tuberosus tuber. In the figure, F stands for fructose, G for glucose, S for sucrose (disaccharide), N for neocestosis (F2-6G1-2F, trisaccharide), I for 1-kestose (G1-2F1-2F, trisaccharide), K for kestosis (G1-2F6-2F, trisaccharide). Higher oligosaccharides (DP 4-9) are also indicated.

Figure 2 shows the TLC analysis of transgenic tobacco plants (KZ) containing the fructosyl transferase gene. express mutans. Oligosaccharides accumulate in these plants. Lane H shows as an control an extract of a Helianthus tuberosus tuber.

Figure 3 shows the SDS-PAGE gel of purified onion seed SST. On this silver stained gel, 6 single band was visible in the SST sample. M stands for molecular weight markers with their size indicated in kilodaltons (kD).

Figure 4 shows the reaction products of purified onion seed SST incubated with sucrose (lanes 4 and 5: Q in vitro). Only trisaccharides are formed. Lane 1 shows the extract of tulip stems (T), lane 2 shows the extract of Helianthus tuberosus tubers (H), lane 3 shows the extract of an Allium cepa bulb (O). M stands for mono-saccharide, S for sucrose (disaccharide), N for neocestose (F2-6G1-2F, trisaccharide), I for 1-kestose (G1-2F1-2F, trisaccharide). Higher oligosaccharides (DP4-5) are also indicated. The products were analyzed on TLC as described by Figure 1.

EXAMPLE 1

Selecting an oen.

1. Naturally occurring genes.

A large number of microbes have been screened for their ability to produce oligosaccharides from sucrose.

For this purpose, bacterial cultures were grown overnight in a liquid nutrient medium. The oligosaccharide-producing activity was determined by incubating a sample of the culture with 200 mH sucrose in the presence of 0.1% Triton X-100. The reaction products were separated by TLC and visualized using a fructose specific reagent (Cairns, A.J. and Pollock, C.J., New Phytol. 109, 399-405 (1988)). As a result of this screening, Streotococcus mutans was found to be an effective oligosaccharide producer (see Figure 1). The oligasaccharide-producing enzymatic activity was purified from the Streptococcus mutans culture by DEAE ion exchanger chromatography and gel permeation chromatography. From this it was found that the enzymatic activity was caused by the product of the ftfen previously described by Shiroza and Kuramitsu, (J. Bacterιοί, 170, 810-816 (1988)).

Subsequently, the fructosyl transferase fftf) gene from plasmid pTS102 (Shiroza and Kuramitsu suoraï was

EcoRV-BglII fragment cloned into the multiple cloning site of pEMBL9 (Dente et al., Nucl. Acids Res. 11, 1645-1655 (1983)) from the lacZ promoter present in this plasmid. transformed coli, enabling the bacterium to produce oligosaccharides.

The production of oligosaccharides was demonstrated by the above mentioned screening method. Untransformed £. coli does not produce oligosaccharides from sucrose.

2. Mutated genes.

By mutagenesis it is possible to adjust the oligosaccharide producing activity of the enzyme as needed. For example, mutations in the gene can be accomplished in the following manner.

For mutagenesis of the ftf-q of Streptococcus mutans, the plasmid pTS102 was integrated into the genome of Svnechococcus sp. PCC 7942 (R2-PIM9) by the genomic integration system (Van der Plas et al., Gene 95, 39-48 (1990)), resulting in support R2-PTS. This cyanobacteria R2-PTS strain expresses the fructosyl transferase gene. The R2-PTS strain is susceptible to sucrose due to polymer accumulation in the periplasm. An R2-PTS culture was mutated with N-methyl-N '-nitro-N-nitrosoguanidine (MNNG) which induces point mutations (T - * C and G - * A mutations). Mutants with altered fructosyl transferase activity were selected. The MNNG mutated culture was plated on sucrose containing medium and a total of 400 sucrose resistant colonies were tested for altered fructosyl transferase activity.

From these colonies, R2-PTS cultures were derived which were concentrated by centrifugation. The pellets thus obtained were resuspended in 50 mM sodium phosphate buffer with 1% Triton X-100, 200 mM sucrose and incubated overnight at 37 ° C. The reaction products were analyzed by TLC analysis (Cairns and Pollock supra). The TLC was developed three times in 85:15 acetone: water and then treated with urea spray as described by Wise et al., Analytical Chemistry 27, 33-36 (1955).

This method preferably stains fructose and fructose-containing polymers.

Of the mutants mainly producing trisaccharides, one was selected to demonstrate in vitro the enzymatic oligosaccharide-forming activity of the mutated ftf-qen as described above.

According to the invention, other mutagenesis methods (site-directed or random) and genes encoding fructosyl transferases from other organisms can also be used to select a gene for a mutant oligosaccharide producing protein.

▼ EXAMPLE 2

Expression of planting in plants.

A. Construction of 35S-ftf-NOS in a plant transformation vector

The plasmid pM0G18, which contains a plant-specific 35S promoter with enhancer duplication and sequences that stimulate the translations of mRNA, has been described by Symons et al. (Bio / Technology 8, 217-221 (1990)). It contains the 35S promoter uidA gene N0S terminator construct. A Stratagene pBluescript II SK plasmid (San Diego, CA, U.S.A.) from which the internal BamHI site had been removed by cutting with BamHI and filling in the overhangs with Klenow and ligating again was used for further cloning. The 35S uidA NOS fragment was obtained by digestion with EcoRI and HindIII of pMOG18 and cloned into this BamHI pBluescript in the corresponding EcoRI / Hin dlll site resulting in plasmid pPA2. Plasmid pPA2 was digested with Ncol and BamHI and the vector containing fragment was isolated.

The fructosyl transferase gene ftf was cloned from the plasmid pTS102 (see above) as an EcoRV / BglII fragment into the multiple cloning site of pEMBL9. The compatible Narrow and BamHI sites were used for this. This resulted in the plasmid pTA12.

In order to obtain an Ncol site near the mature ftfone processing site (nucleotide position 783) (J. Bacteriol. 170, 810-816 (1988)), site-directed mutagenesis was performed as described by Kramer et al. Nucleic Acids Res. 12, 9441-9456 (1984)) with the following oligonucleotide: 5 -GGCTCTCTTCTGTTCCATGGCAGATGAAGC-3 ·. This resulted in piasmid pTD2. As a result, at amino acid position +1 (nucleotide position 783) relative to the mature processing site, a glutamine is changed to a methionine. The NcoI / Psti fragment containing the sequence encoding the mature fructosyl transferase was used for further cloning. From this plasmid, the ftfen was isolated as an NcoI / Pstl fragment and this fragment was ligated into the pPA2 vector containing fragment described above. This results in plasmid pTX. pTX contains the 35S-ftf-NOS framework in which ftf represents the mature fructosyl transferase gene without its signal sequence region. pTX was digested with Xbal and HindlII, the fragment containing the complete construct (35S-ftf-N0S) was cloned into the Xbal / HindiII site of pMOG23 (Symons et al., suprai a derivative of the binary plant vector pBIN19 (Bevan, Nucl Acids Res 12, 8711-8721) This resulted in plasmid pTZ.

B. Manufacture and analysis of transgenic plants expressing the mature ftf gene

The pTZ plasmid was conjugated in Aorobacterium tumefaciens LB4404 (Hoekema et al., Nature 303, 179-180 (1983)) in a three-point cross using the helper plasmid pRK2013 (Lam, Plasmid 13, 200-204 (1985)). .

The construct was introduced in Nicotiana tabacum var. Petit Havanna (SRI) using the leaf blade transformation method (Horsch et al., Science 227, 1229-1232 (1985)).

The regenerated plants were called KP plants and were selected for kanamycin resistance and grown on MS medium (Murashige and Skoog, Physiol. Plant. 15, 473-497 (1962)). Then the plants on earth were grown in the greenhouse and analyzed.

The leaf material was cut and ground in an eppendorf tube. After centrifugation (2 minutes at 16,000 rpm), 1 µl supernatant was analyzed on TLC as described in Example 1.

Oligosaccharides have never been found in wild-type plants or in plants transformed with unrelated constructs. The screening of the transformants using this method showed oligosaccharide accumulative plants (see Figure 2). The expression levels varied between individual plants transformed with the same construct. This is a normal phenomenon in transformation experiments in plants. The variation of the expression levels depends mainly on the integration site in the genome (position effect).

▼ EXAMPLE 3

Producing oil saccharide etc from a plant

In addition to the fructosyl transferase genes used above, which originated from microorganisms, such enzymes are also produced by plants. In this example, the SST gene from onion seed is used.

The onion seed SST protein was purified by chromatographic procedures using the following protocol. The seed was incubated at 22 ° C between damp cloths for 2 to 3 days and homogenized in 50 mM phosphate citrate buffer pH 5.7. The starch and debris were centrifuged at about 10,000 g for 10 minutes. Ammonium sulfate was added to 20% to the supernatant and the precipitate collected by centrifugation. The concentration of ammonium sulfate in the supernatant was increased to 80% and the precipitate collected and dissolved in 20 mM NaAc pH 4.6. The solution was dialyzed overnight with three buffer changes (20 mM NaAc) and the solution clarified by centrifugation. The supernatant was placed on an FPLC monoS column and eluted in 20 inM NaAc pH 4.6 with a 0-0.5 M NaCl gradient. After dialysis against 10 nM NaAc pH 5.6 overnight, the solution was applied to a raffinose epoxysepharose column (Pharmacia) equilibrated in 10 nM NaAc pH 5.6. Elution took place with a linear gradient consisting of 10 mM NaAc pH 5.6 (buffer A) and 10 nM phosphate citrate buffer, pH 7.0, plus 0.5 M NaCl buffer (buffer B). The active fractions were dialyzed overnight against 20 mM phosphate citrate buffer, pH 7.0, and loaded onto a monoQ FPLC column in 20 mM phosphate citrate buffer, pH 7.0. The column was eluted with a gradient of 0-0.5 M NaCl. For final purification, the protein was applied to a Sepharose 6 column and eluted with 50 nM phosphate buffer, pH 6.5, 1% Triton X-100. The silver staining of an SDS-PAGE gel of purified onion seed SST revealed only one band with a molecular weight of about 68,000 d (see Figure 3).

When this purified SST was incubated with sucrose, only 1-kestose was produced. No significant invertase activity was observed (see Figure 4).

The amino acid sequence of the purified protein was determined from peptides obtained by partial degradation. Based on this information, PCR probes were designed to isolate the gene encoding the SST from onion seed. In the same manner as described in Examples 1 and 2, this demonstrated in vitro and in vivo that the gene encodes an enzyme capable of producing oligosaccharides.

EXAMPLE 4

Applicability to other olante species

In order to illustrate the general applicability of the technology, the ftf construct. described in Example 2 introduced in different crops. For example, the potato was transformed according to the method described in Visser, Plant Tissue Culture Manual B5, 1-9,

Kluwer Academie Publishers, 1991. The resulting transgenic plants accumulated oligosaccharides in each organ tested. In addition, the same construct was introduced into the beet (Beta vulqaris L.) which was transformed as described by D'Halluin et al., Biotechnology 10, 309-314 (1992). The resulting transgenic beet plants accumulated significant amounts of oligosaccharides in, for example, their leaves and roots. The same constructs were introduced into Brassica nanus L. which was transformed according to Block et al., Plant Physiol. 91, 694-701 (1989). The resulting transgenic plants accumulated important levels of oligosaccharides in, for example, their leaves and storage organs. It is of course not necessary that the plants are transformed in the indicated ways. Other methods within the reach of those skilled in the art can also be used.

Examples of other plant species that can be modified include but are not limited to corn (Zea mavs L.), wheat (Triticum aestivum L.), barley (Hordeum vulaare L.), rice (Orvza sativa L.), soybean (Glvcin max L.), pea (Pisum sativum L.), bean (Ehass & lMS vvlqgurig L.), chicory (Cichorium intvbus L.), sugarcane (Saccharum officinarum L.), sweet potato (Dioscorea esculenta L.), cassava (Manihot esculenta L .) and grasses (e.g., Lollum spp., £ s & spp., and Festuca spp.).

Plants with natural or induced modified carbohydrate separation patterns may be preferred plants for the introduction of oligosaccharide synthesizing genes. Such plants include but are not limited to natural mutants in starch and sucrose metabolism, and plants in which the starch and sucrose metabolism are modified by molecular and genetic techniques, for example, as described in Sonnewald and Willmitzer, Plant Physiology 99, 1267-1270 , (1992).

EXAMPLE 5

Use of the oil saccharides according to the invention - 'i' '··. *: -

The oligosaccharides produced by the method of the invention can be used as sugar substitutes in various products. Three examples are given below.

1. Ice cream

Ice cream is prepared from the following ingredients: 635 parts of water 90 parts of butterfat 100 parts of skimmed milk powder 170 parts of oligosaccharides of the invention 5 parts of Cremodan SE30 ™ (Grindsted) 0.3 parts of Aspartame ™ flavorings as needed.

The milk powder is dissolved in the water. The whole is heated to 40-45 ° C. The remaining dry ingredients are mixed and dissolved in the warm milk. The melted butter is then added. The whole is then pasteurized at 72 ° C for 10 minutes. The mixture is then homogenized in a two-stage homogenizer at 150/35 bar. The ice cream mixture thus obtained is rapidly cooled to 5 ° C and then the whole is allowed to age for at least 4 hours at 5 ° C. Finally, the ice cream mix is aerated and frozen to an overrun of 100%.

After curing at -35 ° C and storage at -20eC, an ice is obtained, which in taste and texture is similar to ice cream prepared with natural sugars (sucrose, glucose syrup).

2. Muesli bar

A muesli bar was prepared from the following ingredients: 28 parts oligosaccharides according to the invention 68 parts muesli mix 4 parts cocoa

The oligosaccharides were made into a syrup by heating which was mixed with the other ingredients. The bars of the mixture thus obtained were formed in a cylindrical press. Due to the lack of natural sugar, the bar is much lower in calories than the conventional bars.

3. Soda

A soft drink is prepared from the following ingredients: 90 parts of water or fruit juice 8-10 parts of oligosaccharides according to the invention artificial sweetener (s) flavors and colors food acid carbonic acid

All ingredients are dissolved in a portion of the water. The remaining water is then added as carbonated water. The energy value of the soft drink is much less, because no extra natural sugars have been added.

Claims (18)

A method of producing oligosaccharides, comprising the steps of: a) selecting a gene encoding an enzyme capable of converting sucrose into an oligosaccharide; b) linking the gene to appropriate transcription initiation and transcription termination signals to provide an expression construct; c) transforming a suitable plant cell with the expression construct; d) regenerating a transgenic plant from the transformed plant cell; e) cultivating the transgenic plant under conditions that allow for the expression and activity of the enzyme; and f) isolating the oligosaccharides from the transgenic plant. A method according to claim 1, characterized in that the gene encoding an enzyme capable of converting sucrose into an oligosaccharide is of microbial origin. The method according to claim 2, characterized in that the gene encoding an enzyme capable of converting sucrose into an oligosaccharide is the ftf-gene of Streptococcus mutans or a mutated version thereof. A method according to claim 2, characterized in that the gene encoding an enzyme capable of converting sucrose into an oligosaccharide is the SacBen of Bacillus subtilis or a mutated version thereof. A method according to claim 1, characterized in that the gene encoding an enzyme capable of converting sucrose into an oligosaccharide is of vegetable origin. A method according to claim 5, characterized in that the gene encoding an enzyme capable of converting sucrose into an oligosaccharide is a sucrose-sucrose-fructosyl transferase. A method according to claim 6, characterized in that the sucrose-sucrose-fructosyl transferase originates from the onion. A method according to any one of the preceding claims, characterized in that the expression construct further comprises at least one targeting signal sequence. 9. Obtain oligosaccharides by transforming a plai cell with a gene encoding an enzyme capable of converting sucrose into an oligosaccharide; regenerating a transgenic plant from the transformed plant cell; growing the transgenic plant under conditions that allow for expression and activity of the enzyme; and isolating the oligosaccharides from the transgenic plant. Oligosaccharides according to claim 9, characterized by the general formula GBFn, wherein G is glucose and F is fructose and m is equal to 0 or 1 and n is an integer greater than or equal to 0, preferably m is equal to 1 and n ranges from 2 to 8, preferably n equals 2 or 3. A DNA construct for expressing an enzyme capable of converting sucrose into an oligosaccharide in a plant or plant cell, comprising a gene encoding the enzyme, in reading frame linked to plant-specific transcription initiation and termination signals. Transgenic plant cell, comprising the DNA construct according to claim 11. Transgenic plant, to be produced by regeneration from a transgenic plant cell according to claim 12. Transgenic plant tissue from a plant according to claim 13. Use of the oligosaccharide according to claim 9 or 10 as a sugar substitute in foods. Use of the oligosaccharide according to claim 9 or 10 as a dietary fiber in foods. Use of the oligosaceharide according to claim 9 or 10 as a bifidogenic agent in foods. Use of the oligosaccharide according to claim 9 or 10 as a bifidogenic agent in animal feed.